Ribonuclease H of reverse transcriptase and cellular RNases H are related proteins, both structurally and enzymatically. However, they differ in several aspects. Experiments designed to determine the sequences and structures that contribute to the differences between the two types of RNases H have been a focus of some of our project. We have expressed the RNase H domains of HIV and MuLV as separate proteins and studied some of its properties related to binding to RNA-DNA hybrids. E. coli RNase HI is more than 1,000 times as active enzymatically as either of the RNases H derived from the mammalian retroviruses. E. coli RNase HI binds very rapidly to RNA-DNA hybrids as does the MuLV RNase H. Thus, the differences in specific activity between these two enzymes is due to a difference in catalytic activity. In contrast, an E. coli RNase H protein deleted for the region known to be responsible interacting with RNA-DNA substrates (making it similar to HIV RNase H) binds very poorly to its substrate. When we target E. coli RNase H to mammalian retroviruses and yeast retrotransposons, differences in enzymatic properties result in inhibition of viral multiplication for MuLV, TY-1 and possibly HIV. Thus, targeting of cellular RNase H to virus particles may be a useful method for containing retroviral infections.